JPH09122158A - Direct freezing method of pig embryo - Google Patents

Abstract

(57) [Summary] (Modified) [Problem] To establish the cryopreservation of pig embryos. SOLUTION: Ethylene glycol, propylene glycol, serum and textran are added to a pig embryo and frozen in the presence of a basic medium.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a direct freezing method for pig embryos. The present invention also relates to a method for breeding pigs which can be directly transplanted using the frozen pig embryo without ovum examination.

[0002]

2. Description of the Related Art Embryo transplantation of pigs has been attracting attention as a new production technique such as improvement of the growth rate and production of clean pigs, but it has hardly been used for some reasons. The main reason for the impractical application of pig embryo transfer is that the structure of the genital organs is complicated, making it very difficult to perform non-surgical egg collection and transfer, and the embryos are vulnerable to low temperature and cannot be stored. In the breeding area of cattle, a cryopreservation technique that is a semi-permanent preservation method for embryos has already been established, and embryo transfer is widely used as a practical technique. However, let alone freeze preservation of pig embryos,
Since exposure to low temperature reduces viability, pig embryo transfer is subject to time and distance restrictions.

In the first year of the Heisei era, a piglet was born in the world's first cryopreserved embryo transplantation at the Ministry of Agriculture, Forestry and Fisheries (Oguri, 53rd Japan Swine Society: 48-54, 199).
0). Since then, piglets have been born at two domestic institutions, but the success rate to reach the litter is extremely low and the technical level is almost reproducible. Until now, in the conventional stepwise method, only 5 cases were successful in the world and the number of litters was only several (up to 10% per thawed embryo) (Oguri, Ibid., Kameyama et al., 78th Livestock). Breeding Society I-54: 199
0, Kashiwazaki et al., The 78th Japanese Society of Animal Reproduction I-57, 199.
0, Nagashima et al., Theriogenology, 41: 113-118, 199.
4, Nagashima et al., Nature, 374: 416, 1995). Therefore, many embryos have been thawed to remove the antifreezing agent, and the embryos have been selected and transplanted. The stepwise method is also called a serial dilution method. Glycerin or the like is added as a cryoprotectant to freeze the embryo in a straw, and when the embryo is transplanted, the frozen egg is thawed and the fertilized egg is taken out from the straw, and then in 3 to 5 steps. It is a method of transplanting after removing the antifreeze agent in two parts.

[0004]

Unlike bovine embryos, pig embryos do not have a cryopreservation method established, and thus cryopreserved embryos could not be used for the maintenance of lineage pigs and the transportation of pigs. Therefore, the aim is to establish a cryopreservation method for pig embryos.

[0005]

According to the present invention, it has been found that a pig embryo can be directly transplanted to an egg receiving pig by adding dextran, serum, ethylene glycol and propylene glycol to the pig embryo in the presence of a basal medium and freezing. It was

[0006]

The present invention will be described in more detail with reference to the following examples.

[0007]

Example 1 As a test embryo, an expanded blastocyst stage embryo collected 6 days after estrus from a pig that had been artificially fertilized during estrus was used. Supplement calf serum (serum prepared by adding various growth factors and trace elements to a cryoprotectant. In this example, Gib)
The composition of the frozen solution for direct transplantation, to which Calf Supreme ^™ manufactured by Co. was used) and dextran were added, is as follows. 4% (v / v) ethylene glycol 4% (v / v) propylene glycol 20% (v / v) supplement calf serum 1% (w / v) dextran Basal medium: TCM199 Freezing container: 0.5 ml plastic straw Insertion method into straw: As shown in FIG. 1, the straw was filled in the following order from the cotton plug side. 1. Freezing solution 2. Air 3. Freezing solution and embryo 4. Air 5. Frozen Solution Program Freezer: Tokyo Rika Co., Ltd. Ice: -6 ° C Cooling temperature: 0.5 ° C / min Liquid nitrogen input temperature: -32.5 ° C Melting method: Remove straw from liquid nitrogen, and air (room temperature ) For 5-10 seconds and melt in hot water at 35 ° C. Transplantation method: The entire contents of the straw were placed in a culture dish, and the embryo was set in a catheter as it was and transplanted. Table 1 shows a comparison between the present invention and the conventional method. Table 1 ──────────────────────────────────── Test section Frozen solution Thaw method ────── ────────────────────────────── Conventional method 10% Glycerol 5 steps (6.7%, 3.3 (stepwise method) ) + 20% supplement%, 0% glycerol + 0. Bovine serum (SCS) or 3M sucrose + 20% fetal calf serum (FCS) + SCS or 0.2M, 0.1% dextran 1M sucrose + 20% basal medium PBS or SCS or FCS TCM199 method of the present invention 4% ethylene glycol direct transplantation method (direct method) + 4% propylene glycol + 20% supplement calf serum + 1% dextran basal medium TCM199 ───────────── ────── ───────────────── The above results are shown in Table 2. Table 2 Results of transplantation of frozen pig embryos ──────────────── ───────────────────── Number Number of test sections Number of thawed embryos Number of transplanted embryos Number of deliveries Remarks (% thawed embryos) <% transplanted embryos> (% thawed embryos) ── ────────────────────────────────── 1 SW-1 37 20 (54%) 4 heads <20%> (11%) Stillbirth 1 Mummy 1 2 SW-2 50 20 (38%) 5 heads <25%> (5%) 3 SW-3 35 20 (57%) 0 heads <0%> (0%) Not conception 4 Direct method 20 20 (100%) 10 heads <50%> (50%) Stillbirth 5 SW-3 49 20 (41%) 2 heads <10%> (4%) 6 Direct method 20 20 (100%) 1 Head <5%> (5%) ──────────────────────────────────── Note: SW-1 Basal medium PBS, FCS, lecithin added SW-2 Basal medium PBS, FCS, tocopherol added SW-3 Basal medium TCM199, SCS Direct method (after thawing the embryo, the solution is transferred as it is to a catheter for transplantation and transplanted) The method of the present invention.

[Brief description of the drawings]

FIG. 1 shows a cross-sectional view of a straw containing an embryo.

[Explanation of symbols]

 The straw body b Cotton plug C Frozen solution N Air air Frozen solution + embryo F seal

Claims (4)

[Claims] 1. A freezing solution containing ethylene glycol, propylene glycol, serum and dextran is added to a pig embryo in the presence of a basal medium, followed by freezing.
Direct freezing method for pig embryos. 2. The concentration of dextran is about 1%,
The method of claim 1. 3. The method according to claim 1, wherein the serum concentration is about 20%. 4. A frozen solution prepared by adding serum to a cryoprotectant selected from the group consisting of ethylene glycol, propylene glycol and dextran, and a pig embryo are frozen together and stored, and the frozen embryo is thawed at the time of use, and as it is. A method for breeding pigs, which comprises transplanting the pigs into a receiving pig.